EP3380462B1 - Verfahren zur analyse und auflösung von präparaten von dianhydrogalactitol und derivaten oder analoga davon - Google Patents

Verfahren zur analyse und auflösung von präparaten von dianhydrogalactitol und derivaten oder analoga davon Download PDF

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EP3380462B1
EP3380462B1 EP16869177.2A EP16869177A EP3380462B1 EP 3380462 B1 EP3380462 B1 EP 3380462B1 EP 16869177 A EP16869177 A EP 16869177A EP 3380462 B1 EP3380462 B1 EP 3380462B1
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dianhydrogalactitol
dulcitol
acetonitrile
rrt
water
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EP3380462A4 (de
EP3380462A1 (de
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Qiang Xu
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Del Mar Pharmaceuticals BC Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N30/22—Injection in high pressure liquid systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
    • C07D303/14—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by free hydroxyl radicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/30—Partition chromatography
    • B01D15/305—Hydrophilic interaction chromatography [HILIC]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8872—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample impurities
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph

Definitions

  • This invention is directed to improved analytical methods for dianhydrogalactitol and derivatives or analogs thereof, especially involving high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • Dianhydrogalactitol (1,2:5,6 dianhydrogalactitol or DAG) is one of a number of hexitols or hexitol derivatives having significant pharmacological activity, including chemotherapeutic activity.
  • dianhydrogalactitol has been suggested for use in chemotherapy, such as in United States Patent No. 7,157,079 to Nielsen et al.
  • Dianhydrogalactitol has activity against a number of neoplasms.
  • dianhydrogalactitol is to be used successfully as a therapeutic agent, an extremely high degree of purity and the removal of impurities is essential. The presence of impurities can lead to undesirable side effects.
  • impurities present in a batch of the amino acid tryptophan, a normal constituent of protein were responsible for a significant outbreak of eosinophilia-myalgia syndrome, which caused a large number of cases of permanent disability and at least 37 deaths.
  • the therapeutic agent such as dianhydrogalactitol is to be employed in patients with compromised immune systems or liver or kidney dysfunction, or in elderly patients. Such patients may experience a greater incidence of undesirable side effects owing to their sensitivity to contaminants.
  • One of the impurities found in preparations of dianhydrogalactitol is dulcitol. Other impurities exist in preparations of dianhydrogalactitol as well, depending on their method of preparation.
  • US2014/315318 A1 discloses an analytical method for analyzing the presence and quantity of impurities present in a preparation of dianhydrogalactitol.
  • WO2013/128285 A2 discloses an analytical method for analyzing the presence and quantity of impurities present in a preparation of dianhydrogalactitol.
  • the present invention is defined by the claims.
  • An improved analytical method for determining the purity of dianhydrogalactitol and derivatives or analogs thereof and detecting impurities and degradation products in preparations of dianhydrogalactitol and derivatives or analogs thereof that meets these needs is described herein.
  • the analytical method described herein is suitable for determining the purity of dianhydrogalactitol and detecting impurities and degradation products in preparations of dianhydrogalactitol.
  • HPLC high performance liquid chromatography
  • ELSD evaporative light scattering detection
  • an analytical method can detect the impurities dulcitol and dibromodulcitol in a preparation of dianhydrogalactitol.
  • One aspect of the present invention is an analytical method for analyzing the presence and quantity of impurities present in a preparation of dianhydrogalactitol comprising the steps of:
  • the method detects and quantitates at least one of: (1) dulcitol; (2) dibromodulcitol; (3) an impurity other than dulcitol; and (4) a degradation product of dianhydrogalactitol.
  • the flow rate is from about 0.72 mL/min to about 0.88 mL/min.
  • the flow rate is about 0.80 mL/min.
  • the scheme for elution is (1) 0.00 minutes, 90% of 100% acetonitrile; 10% of 100% water; (2) 3.00 minutes, 90% of 100% acetonitrile; 10% of 100% water; (3) 20.00 minutes, 75% of 100% acetonitrile, 25% of 100% water; (4) 25.00 minutes, 50% of 100% acetonitrile, 50% of 100% water; (5) 30.00 minutes, 50% of 100% acetonitrile, 50% of 100% water; (6) 30.10 minutes, 90% of 100% acetonitrile, 10% of 100% water; and (7) 40.00 minutes, 90% of 100% acetonitrile, 10% of 100% water.
  • the injection volume is from about 15 ⁇ L to about 35 ⁇ L.
  • the injection volume is about 25 ⁇ L.
  • the column temperature is from about 28° C to about 32° C.
  • the column temperature is about 30° C.
  • the run time is about 40 minutes.
  • the injector temperature is at about 5° C. In another alternative, the injector temperature is at about 20° C to about 22° C.
  • the method can include a needle wash with 100% acetonitrile.
  • the evaporative light scattering detection comprises post-column addition of a volatile solvent to enhance evaporation of the aqueous fraction of any mobile phase present.
  • the volatile solvent can be selected from the group consisting of methanol, ethanol, isopropanol, and acetonitrile.
  • the ELSD detection conditions are a temperature from about 36° C to about 44° C, a gas pressure from about 2.0 bar to about 3.0 bar, and a gain of 7.
  • the ELSD conditions are a temperature from about 38° C to about 42° C, a gas pressure from about 2.25 bar to about 2.75 bar, and a gain of 7. In a still more preferred alternative, the ELSD conditions are a temperature of about 40° C, a gas pressure of about 2.5 bar, and a gain of 7.
  • the evaporative light scattering detection can be compatible with electrospray LC/MS. When the evaporative light scattering detection is compatible with electrospray LC/MS, an electrospray tandem mass spectrometer can be installed and connected on-line to an HPLC system with ELSD.
  • tandem mass spectral data providing chemical information for each of the impurities and degradation products that may be present in a preparation of dianhydrogalactitol can be collected.
  • Mass spectroscopy in tandem with HPLC can provide molecular ion information and possible chemical structures having a molecular weight consistent with the molecular ion information for each of the observed impurities and degradation products.
  • at least one impurity or degradation product is identified by separation by column chromatography followed by at least one purification procedure to yield a solid unknown sample.
  • the solid unknown sample can be characterized for identification by at least one standard analytical procedure selected from the group consisting of nuclear magnetic resonance (NMR), mass spectroscopy (MS), Fourier transform infrared spectroscopy (FT-IR), elemental analysis, determination of purity by HPLC, and determination of water content by the Karl Fischer titration method.
  • NMR nuclear magnetic resonance
  • MS mass spectroscopy
  • FT-IR Fourier transform infrared spectroscopy
  • elemental analysis determination of purity by HPLC
  • determination of water content by the Karl Fischer titration method Karl Fischer titration method.
  • the method can further comprise the step of performing preparative HPLC collection of at least one specific substance peak present in a preparation of dianhydrogalactitol.
  • the at least one specific substance peak present in the preparation of dianhydrogalactitol can be an impurity.
  • the at least one specific substance peak present in the preparation of dianhydrogalactitol can be a degradation product.
  • This invention is directed to improved analytical methods for determining the purity of dianhydrogalactitol and derivatives or analogs thereof and determining the existence and concentration of impurities present in preparations of dianhydrogalactitol and derivatives or analogs thereof.
  • An improved analytical method for determining the purity of dianhydrogalactitol and derivatives or analogs thereof and detecting impurities and degradation products in preparations of dianhydrogalactitol and derivatives or analogs thereof that meets these needs is described herein.
  • the analytical method is suitable for determining the purity of dianhydrogalactitol and detecting impurities and degradation products in preparations of dianhydrogalactitol.
  • dulcitol One of the significant impurities present in dianhydrogalactitol preparations is dulcitol.
  • the structure of dulcitol is shown below in Formula (II).
  • dibromodulcitol Yet another impurity that can be present in dianhydrogalactitol preparations is dibromodulcitol.
  • the structure of dibromodulcitol is shown below in Formula (III).
  • impurities can be present in preparations of dianhydrogalactitol.
  • the exact identity of such impurities can depend on the routes of synthesis and purification for dianhydrogalactitol.
  • Such impurities can also represent degradation products of dianhydrogalactitol.
  • HPLC high performance liquid chromatography
  • ELSD evaporative light scattering detection
  • MS mass spectroscopy
  • HPLC is performed by employing a reverse phase amide column.
  • a particularly preferred amide column is a 4.6 mm ⁇ 250 mm column employing amide chemistry in a hydrophilic interaction mode; the column employs ethylene bridged hybrid (BEH) technology.
  • the particles of the column are of spherical shape and have a particle size of 3.5 ⁇ m.
  • the particles have a hybrid substrate and have a pore size of 130 ⁇ .
  • a particularly preferred reverse phase amide column is a Waters XBridge BEH amide column No. 186004870 (Waters, Milford MA). Other reverse phase amide columns are known in the art.
  • a preferred alternative is to use 100% acetonitrile as Mobile Phase A and 100% water as Mobile Phase B.
  • a preferred flow rate is from about 0.72 mL/min to about 0.88 mL/min; a particularly preferred flow rate is about 0.80 mL min.
  • a preferred scheme for elution is as follows: (1) 0.00 minutes, 90% of 100% acetonitrile; 10% of 100% water; (2) 3.00 minutes, 90% of 100% acetonitrile; 10% of 100% water; (3) 20.00 minutes, 75% of 100% acetonitrile, 25% of 100% water; (4) 25.00 minutes, 50% of 100% acetonitrile, 50% of 100% water; (5) 30.00 minutes, 50% of 100% acetonitrile, 50% of 100% water; (6) 30.10 minutes, 90% of 100% acetonitrile, 10% of 100% water; and (7) 40.00 minutes, 90% of 100% acetonitrile, 10% of 100% water.
  • the injection volume can be from about 15 ⁇ L to about 35 ⁇ L.
  • a preferred injection volume is 25 ⁇ L.
  • the column temperature can be from about 28° C to about 32° C.
  • a preferred column temperature is about 30° C.
  • a preferred run time is about 40 minutes.
  • the injector temperature can be at about 5° C or at about room temperature (about 20°C to about 22° C).
  • the analytical method can include a needle wash with 100% acetonitrile.
  • ELSD evaporative light scattering
  • An evaporative light scattering detector (ELSD) atomizes the column eluate, shines light on the resulting particulate components, and detects the resulting scattered light. Theoretically, an ELSD can detect any nonvolatile component.
  • the evaporative light scattering detection of a non-chromogenic compound is based on nebulization of the HPLC eluent and evaporation of mobile-phase solvents to produce atomizing solute particles for light scattering detection. This nebulization and solvent evaporation process to produce atomizing analyte solute particles is comparable to the electrospray LC/MS procedure.
  • the ELSD detection is compatible with electrospray LC/MS.
  • Implementation of an HPLC method with ELSD detection that is compatible with electrospray LC/MS application typically involves post-column addition of a volatile solvent to enhance evaporation of the aqueous fraction of any mobile phase present.
  • the volatile solvent is typically selected from the group consisting of methanol, ethanol, isopropanol, and acetonitrile.
  • the ELSD detection conditions are a temperature from about 36° C to about 44° C, a gas pressure from about 2.0 bar to about 3.0 bar, and a gain of 7.
  • the ELSD conditions are a temperature from about 38° C to about 42° C, a gas pressure from about 2.25 bar to about 2.75 bar, and a gain of 7. More preferably, the ELSD conditions are a temperature of about 40° C, a gas pressure of about 2.5 bar, and a gain of 7.
  • an electrospray tandem mass spectrometer can be installed and connected on-line to an HPLC system with ELSD.
  • Mass spectral data providing molecular information and tandem mass spectral data providing chemical structural information for each of the impurities that may be present in a preparation of dianhydrogalactitol can be collected.
  • Mass spectroscopy in tandem with HPLC will provide molecular ion information and possible chemical structures having a molecular weight consistent with the molecular ion information for each of the observed impurities and degradation products.
  • one analytical method for analyzing the presence and quantity of impurities present in a preparation of dianhydrogalactitol comprises the steps of:
  • the compounds other than dianhydrogalactitol itself can be at least one of: (1) dulcitol; (2) dibromodulcitol; (3) an impurity other than dulcitol; and (4) a degradation product of dianhydrogalactitol.
  • Suitable conditions for reversed phase chromatographic analysis and ELSD detection are as described above.
  • a method according to the present invention further comprises the step of analyzing one or more peaks eluting from the high performance liquid chromatography by electrospray tandem mass spectroscopy.
  • a method according to the present invention further comprises the step of preparative HPLC collection of at least one specific dianhydrogalactitol-related substance peak.
  • the unknown impurity or degradation product can be identified by separation by column chromatography followed by at least one purification procedure to yield a solid unknown sample which can then be characterized for identification by at least one standard analytical procedure selected from the group consisting of nuclear magnetic resonance (NMR), mass spectroscopy (MS), Fourier transform infrared spectroscopy (FT-IR), elemental analysis, determination of purity by HPLC, and determination of water content by the Karl Fischer titration method. These methods are well known in the art.
  • the method can further comprise the step of performing preparative HPLC collection of at least one specific substance peak present in a preparation of dianhydrogalactitol.
  • the at least one specific substance peak present in the preparation of dianhydrogalactitol can be an impurity.
  • the at least one specific substance peak present in the preparation of dianhydrogalactitol can be a degradation product.
  • Example 2 The invention is illustrated by the following Example. This Example is for illustrative purposes only, and is not intended to limit the invention.
  • this test procedure is used for determining one or more related substance or substances in a preparation of dianhydrogalactitol (DAG) drug substance and drug product by high performance liquid chromatography (HPLC) with ELSD detection (HPLC-ELSD).
  • HPLC high performance liquid chromatography
  • HPLC-ELSD ELSD detection
  • a reversed phase column Waters Amide, 3.5 ⁇ m, 4.6 ⁇ 250 mm
  • ACN acetonitrile
  • the flow rate is 0.8 mg/ml.
  • the peak of DAG is detected with a retention time at about 5.20 min.
  • the DAG samples are prepared at target concentration of 8 mg/mL.
  • Reagents and reference standards include: (1) purified water (H 2 O), HPLC grade or equivalent; (2) acetonitrile (ACN), HPLC grade or equivalent; and (3) dulcitol reference standard.
  • Instruments and apparatus include: (1) Shimadzu LC20A HPLC system or equivalent equipped with ELSD detector; (2) Waters Amide reverse phase HPLC column, 3.5 ⁇ m, 4.6 ⁇ 250 mm, PN: 186004870); (3) Purity water generator; and (4) analytical balance.
  • the volumes may be scaled up or down as appropriate for testing.
  • a diluent with acetonitrile/water (85:15, v/v) can be prepared by thoroughly mixing 850 mL of ACN and 150 mL of water.
  • the mobile phases are 100% ACN (Mobile Phase A) and 100% water (Mobile Phase B).
  • Dulcitol linearity standard solutions of 0.016 mg/mL (0.2%), 0.08 mg/mL (1%), and 0.4 mg/mL (5%) are prepared.
  • the weights and volumes given are the recommended amounts for routine quantitative analysis. Alternative amounts may be used, provided that the final concentrations remain the same.
  • the glassware is to be pre-rinsed five times with purified water and ACN to minimize potential contamination.
  • concentrations can be prepared.
  • a dulcitol concentration standard of 3% (0.24 mg/mL)
  • accurately weigh about 24 mg of dulcitol reference standard into a 100 mL volumetric flask add 15 mL purified water and sonicate to dissolve, dilute to volume with acetonitrile and mix well. Label them as L-3%-#1 (STD#1) and L-3%-#2 (STD#2).
  • the volumetric flask should be cleaned completely by rinsing with purified water and ACN before use.
  • One replicate can be prepared for 0.08 mg/mL (1%). For example, accurately weigh about 20 mg of dulcitol reference standard into a 250 mL volumetric flask, add 37.5 mL purified water and sonicate to dissolve, dilute to volume with ACN and mix well. Label it as L-1%.
  • the standard solutions can be stored up to 48 hrs at 2-8° C and can be stored up to 52 hrs at ambient laboratory condition.
  • the standard solutions can be used to generate a standard curve as described further below.
  • a dulcitol sensitivity solution (0.008 mg/mL (0.1%) in one replicate can be prepared as shown below in Table 2.
  • Table 2 Levels Example Dilution Procedure Label AS Target Concentration (mg/mL) 0.1% 1 mL L-5%-#1 ⁇ 50 mL SS 0.008
  • a dianhydrogalactitol (DAG) standard solution (8 mg/mL) (for identification purposes only) can be prepared as follows: Accurately weigh, in one replicate, about 80 mg of DAG working standard into clean 10 mL volumetric flasks. Dissolve DAG working standard in approximately 5 mL of diluent, diluted to volume with diluent, and mix. The solution can be liquored and stored frozen for future use until the relevant peaks are no longer detectable in the chromatogram.
  • the test sample solutions are prepared as follows.
  • the sample preparation for DAG drug substance or API can be as follows. Accurately weigh, in duplicate, about 80 mg of DAG API sample into clean 10 mL volumetric flasks. Dissolve DAG API sample in approximately 5 mL of diluent, diluted to volume with diluent, and mix.
  • the sample preparation for DAG drug product (lyophilized powder, 40 mg/vial) can be as follows: Clean and remove the closure of the vial. Reconstitute the lyophilized vial with 5.0 mL diluent to yield an 8 mg/mL solution. Prepare samples in duplicate (using 2 vials). The sample solutions can be stored up to 48 hrs at 2-8° C and 52 hrs at ambient laboratory conditions.
  • the HPLC column is to be conditioned at initial method conditions and equilibrated prior to the analysis of samples.
  • the sequence is bracketed by injecting STD#1 (L-3%#1) once, followed by no more than 6 injections of sample solution and, at the end of the sequence, again injecting STD#1 (L-3%#1).
  • the interference should be not more than the dulcitol peak in the sensitivity solution.
  • the signal to noise (S/N) of dulcitol peak in the sensitivity solution should be no less than 10.
  • the correlation of the logarithmic calibration curve for L-0.2%, L-1% and L-3%#1 (first injection) should be no less than 0.980.
  • the relative standard deviation (RSD) of Lg (peak area) of dulcitol for the first five initial injections of STD#1 (L-3%#1) should be ⁇ 5%.
  • the tailing factor of dulcitol peak in the first injection of STD#1 (L-3%#1) should be ⁇ 2.0.
  • the recovery of STD#2 (L-3%#2) should be within 95%-105%.
  • C STD#1 is the concentration of dulcitol reference standard in standard solution #1 (STD#1) (mg/mL)
  • C STD#2 is the concentration of dulcitol reference standard in standard solution #2 (STD#2) (mg/mL)
  • a STD#1 is the average peak area of dulcitol in five initial injections of standard solution #1 (STD#1)
  • a STD#2 is the average peak area of dulcitol in five initial injections of standard solution #2 (STD#2).
  • the ratio between the Lg(peak area) of bracketing injection and the average Lg(peak area) of first five injections of STD#1 (L-3%#1) should be within 95%-105%.
  • the signal to noise (S/N) should be no less than 3.
  • the determination of the detection limit (LOD) for dulcitol, one of the expected impurities in a preparation of dianhydrogalactitol, is shown in Figure 2.
  • Figure 2 is a graph showing the results of chromatography of an 0.004 mg/mL dulcitol solution (0.05% of the nominal concentration) to determine the detection limit (LOD) for dulcitol.
  • Figure 3 is a graph showing the results of chromatography of an 0.008 mg/mL dulcitol solution (0.1% of the nominal concentration) to determine the quantitation limit (LOQ) for dulcitol.
  • the RSD% should be recorded. For 0.5% and above, the RSD% should be ⁇ 20%. The combined RSD% should be ⁇ 20%.
  • the absolute difference should be no more than 0.2% for an impurity at ⁇ 1.0%.
  • the relative difference should be controlled within ⁇ 20%. No new impurities ⁇ 0.1% should be observed.
  • the recovery of the aged solutions should be within 85%-115% of the initial assay.
  • the absolute difference should be no more than 0.2% for an impurity at ⁇ 1.0%.
  • the result of the aged solution should be within 80%-120% of the original. No new impurities at ⁇ 1.0% should be observed.
  • a STD is represents the area of dulcitol for Linearity Standard Solution (for L-3%, the first injections of STD#1 can be used);
  • V STD is the dilution volume for Linearity Standard Solution (mL);
  • W STD is the dulcitol weight of Linearity Standard Solution (mg);
  • P is the purity (or content) of dulcitol;
  • b is the Y-intercept of the established calibration curve; and k is the slope of the established calibration curve.
  • a SPL is the area of individual related substance
  • V SPL is the dilution volume of sample solution
  • b is the Y-intercept of the established calibration curve
  • k is the slope of the established calibration curve
  • W SPL is the DAG drug substance weight (mg).
  • the results of a linearity test using dulcitol are shown in Figure 4 ; these results are from Table 18, below.
  • the calculation for total related substances should sum all individual related substances not less than 0.1%. Void and blank peaks should not be added to the total related substances.
  • Table 5 Composition Unit Formula Qty (mg)/Vial w/w, % DAG for Injection 40 100 Total 40 100
  • the RSD% are shown in Tables 7 (Run 1) and Table 8 (Run 2), below, for two duplicate runs.
  • P-0.5% contaminants present at the 0.5% level or higher
  • dulcitol is shown in Table 9, below; in Table 9, dulcitol was shown to be present at a level of 0.59%.
  • the combined RSD% for intermediate precision (a measure of precision under a defined set of conditions: same measurement procedure, same measuring system, same location, and replicate measurements on the same or similar objects over an extended period of time) for the level of 0.5% is shown in Table 10, below.
  • the absolute differences for ELSD gas pressures of 2.6 bar and 2.4 bar are shown in Table 11, below. No impurity at >1.0% was observed, and no new impurity at ⁇ 0.1% was observed.
  • Results for ELSD temperature of 39° C and 41° C are shown in Table 12, below. No impurity at >1.0% was observed, and no new impurity at ⁇ 0.1% was observed.
  • a dulcitol standard solution was prepared at 1.0% of DAG nominal conc. in one replicate and placed the solution under ambient laboratory conditions and refrigerated conditions (2-8° C), respectively, using 1% Linearity standard solutions).
  • the solution was reanalyzed at different time intervals, such as 2 hours, 4 hours, 8 hours, 24 hours and 48 hours. For time points of less than 24 hours, solution stability is evaluated against fresh standards. The recovery of the aged solutions is evaluated.
  • sample solutions a sample solution at a specified DAG nominal concentration was prepared in one replicate and placed under laboratory conditions at ambient temperature or under refrigerated conditions (2-8° C). The solution was reanalyzed at different time intervals, such as 2 hours, 4 hours, 8 hours, 24 hours and 48 hours. For time points of less than 24 hours, solution stability is evaluated against fresh standards.
  • Figure 1 is a graph showing overlaid chromatograms of blank, a fresh tablet sample, a standard solution, and a mock sample solution (DAG is dianhydrogalactitol and DBD is dibromodulcitol).
  • DAG is dianhydrogalactitol
  • DBD is dibromodulcitol
  • Figure 5 is a graph showing impurities in a sample solution.
  • the impurities include impurities at relative retention times (RRTs) of 0.48, 0.51, 0.56, 0.71, 1.02, 1.07, 1.19, 1.23, and 1.27.
  • RRTs relative retention times
  • Table 18 Level Actual Conc. (mg/mL) Ig(Actual Conc.) Peak Area Ig(Peak Area) Slope Y-intercept Correl (r) 0.1% 0.00797 -2.0985 11663 4.0668 1.7212 7.7258 0.9985 0.5% 0.03985 -1.3996 236370 5.3736 1.0% 0.07970 -1.0985 774592 5.8891 2.0% 0.15940 -0.7975 2238583 6.3500 3.0% 0.23910 -0.6214 3939708 6.5955
  • Table 19 shows the accuracy results for dulcitol.
  • Table 20 shows the precision (repeatability) results for Run 1.
  • Table 20 Peak Name Content (%, #1) Content (%, #2) Content (%, #3) Content (%, #4) Content (%, #5) Content (%, #6)
  • RRT 0.56 0.27 0.28 0.27 0.28 0.28 0.28 0.28 1.0
  • RRT 1.02 0.09 0.08 0.09 0.08 0.08 0.08 0.08 0.08 5.2 RRT 1.07 0.08 0.08 0.09 0.08 0.08 0.08 0.08 0.08 4.0
  • Table 22 shows the precision (repeatability) results at P-0.5%.
  • Table 23 shows the precision results (intermediate precision) for dulcitol.
  • Table 24 Sample Name Content% 40°C Content% 39 °C Content% 41°C Absolute Difference between 39°C and 40°C Absolute Difference between 41°C and 40°C RRT0.48 0.1787 0.1704 0.1795 0.01 0.001 RRT0.51 0.3164 0.3113 0.3163 0.01 0.0001 RRT0.56 0.3021 0.2992 0.3096 0.003 0.01 RRT0.71 0.8608 0.8961 0.8724 0.04 0.01 Dulcitol 0.1814 0.1660 0.1789 0.02 0.002 RRT1.02 0.1037 0.0932 0.1044 0.01 0.001 RRT1.07 0.1094 0.1005 0.1197 0.01 0.01 0.01
  • Table 25 shows the results of a study with variation of ELSD gas pressure.
  • Table 25 Sample Name Content% 2.5bar Content% 2.6bar Content% 2.4bar Absolute Difference between 2.6bar and 2.5bar Absolute Difference between 2.4bar and 2.5bar RRT0.48 0.2041 0.2051 0.2092 0.001 0.01 RRT0.51 0.2511 0.2616 0.2651 0.01 0.01 RRT0.56 0.2626 0.2687 0.2694 0.01 0.01 RRT0.71 0.8570 0.8730 0.8771 0.02 0.02 Dulcitol 0.1591 0.1631 0.1578 0.004 0.001
  • Table 26 shows the results of standard solution stability at 2-8° C for Run 1.
  • Table 26 Time point Concentration (mg/mL) Assay(%) Recovery (v.s. Initial, % ) 0h 0.0895 109.233 N/A 2h 0.0894 109.076 100 4h 0.0895 109.164 100 8h 0.0888 108.302 99 25h 0.0894 109.056 100 48h 0.0893 108.947 100
  • Table 27 shows the results of standard solution stability at 2-8° C for Run 2.
  • Table 27 Sample Name Content(%) Absolute difference (%) 0h 2h 4h 8h 25h 48h 2h 4h 8h 25h 48h RRT 0.48 0.15 0.16 0.15 0.16 0.18 0.17 0.01 0.004 0.01 0.03 0.02
  • RRT 0.56 0.37 0.36 0.36 0.36 0.36 0.38 0.38 0.01 0.004 0.01 0.02 0.01
  • Table 28 shows the results of standard solution stability at ambient laboratory conditions for Run 1.
  • Table 28 Time point Concentration (mg/mL) Assay(%) Recovery (v.s. Initial, % ) 0h 0.0898 109.944 N/A 2h 0.0897 109.778 100 4h 0.0891 109.059 99 8h 0.0887 108.590 99 25h 0.0903 110.553 101 48h 0.0891 108.986 99
  • Table 29 Sample Name Content(%) Absolute difference (%) 0h 2h 4h 8h 27h 52h 2h 4h 8h 27h 52h RRT 0.48 0.15 0.16 0.17 0.18 0.25 0.29 0.002 0.01 0.02 0.1 0.1 RRT 0.51 0.32 0.31 0.32 0.32 0.34 0.32 0.01 0.001 0.002 0.02 0.005 RRT 0.56 0.36 0.35 0.37 0.36 0.37 0.36 0.01 0.01 0.01 0.0004 0.01 0.001 Dulcitol 0.23 0.24 0.24 0.25 0.25 0.24 0.004 0.01 0.01 0.02 0.01 RRT 1.02 0.11 0.12 0.12 0.12 0.13 0.13 0.01 0.01 0.003 0.02 0.01 RRT 1.07 0.15 0.14 0.14 0.15 0.18 0.15 0.002 0.01 0.0005 0.03 0.001 RRT 1.19 0.10 0.09 0.09 0.09 0.10 0.10 0.01 0.01 0.01 0.001 0.01 RRT
  • the system needs to be clean and stable and no interference peak is to be observed in the blank chromatograms at the retention time of dulcitol. If any interference exists, the interference should be not more than the dulcitol peak in the sensitivity solution.
  • the signal to noise (S/N) of dulcitol peak in sensitivity solution should be no less than 10.
  • the correlation of the logarithmic calibration curve for L-0.2%, L-1% and L-5%#1 (first injection) should be no less than 0.980.
  • the relative standard deviation (RSD) of Lg (peak area) of dulcitol for the first five initial injections of STD#1 (L-5%#1) should be ⁇ 5%.
  • the tailing factor of the dulcitol peak in the first injection of STD#1 (L-5%#1) should be ⁇ 2.0.
  • the recovery of STD#2 (L-5%#2) should be within 95%-105%.
  • the ratio between the Lg(peak area) of bracketing injection and the average Lg(peak area) of first five injections of STD#1 (L-5%#1) should be within 95%-105%.
  • the system needs to be clean and stable and no interference peak is to be observed in the blank chromatograms at the retention time of dulcitol. If any interference exists, the interference should be not more than the dulcitol peak in the sensitivity solution.
  • the signal to noise (S/N) of dulcitol peak in sensitivity solution should be no less than 10.
  • the correlation of the logarithmic calibration curve for L-0.2%, L-1% and L-3%#1 (first injection) should be no less than 0.980.
  • the RSD of Lg (peak area) of dulcitol for the first five initial injections of STD#1 (L-3%#1) should be ⁇ 5%.
  • the tailing factor of dulcitol peak in the first injection of STD#1 (L-3%#1) should be ⁇ 2.0.
  • the recovery of STD#2 (L-3%#2) should be within 95%-105%.
  • the ratio between the Lg(peak area) of bracketing injection and the average Lg(peak area) of first five injections of STD#1 (L-5%#1) should be within 95%-105%.
  • the signal to noise (S/N) should be not less than 3.
  • the signal to noise (S/N) should be not less than 10.
  • the correlation coefficient (r) is to be no less than 0.980.
  • the relative standard deviation should be ⁇ 20%.
  • the combined RSD% should be ⁇ 20%.
  • the absolute difference should be not more than 0.2% for an impurity ⁇ 1.0%.
  • the relative difference should be controlled within ⁇ 20%. No new impurities should be observed.
  • the recovery of the aged solutions should be within 85%-115% of the initial assay.
  • the absolute difference should be not more than 0.2% for an impurity ⁇ 1.0%.
  • the result of the aged solution should be within 80%-120% of the initial result. No new impurities ⁇ 0.1% should be observed.
  • the analytical system and method described in this example meets all criteria for sensitivity, precision, robustness, stability, and signal to noise ratio, including the signal to noise ratio at the detection limit. Accordingly, this analytical system and method is useful for the analysis of dianhydrogalactitol and impurities found in preparations of dianhydrogalactitol, including dulcitol, dibromodulcitol, and other impurities.
  • Methods according to the present invention possess industrial applicability for analysis of dianhydrogalactitol preparations and determination and quantitation of impurities in dianhydrogalactitol preparations.
  • the invention encompasses each intervening value between the upper and lower limits of the range to at least a tenth of the lower limit's unit, unless the context clearly indicates otherwise. Moreover, the invention encompasses any other stated intervening values and ranges including either or both of the upper and lower limits of the range, unless specifically excluded from the stated range.

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Claims (15)

  1. Ein analytisches Verfahren zum Analysieren des Vorhandenseins und der Menge von in einer Zubereitung von Dianhydrogalactitol vorhandenen Unreinheiten, das die folgenden Schritte beinhaltet:
    a) Analysieren einer Zubereitung von Dianhydrogalactitol durch Unterziehen der Zubereitung einer Hochdruckflüssigkeitschromatographie unter Verwendung einer Umkehrphasen-Amidsäule, die Amidchemie in einem hydrophilen Interaktionsmodus und Bridged-Ethylen-Hybrid-Technologie (BEH) mit Elution mit einem Mobile-Phase-Gradienten anwendet, wobei eine erste mobile Phase 100%iges Acetonitril ist und eine zweite mobile Phase 100%iges Wasser ist, um Dianhydrogalactitol von Dulcitol und anderen Verunreinigungen der Zubereitung zu trennen; und
    b) Bestimmen der relativen Konzentration von einem oder mehreren durch Hochdruckflüssigkeitschromatographie aufgelösten Peaks, die andere Verbindungen als Dianhydrogalactitol selbst darstellen.
  2. Verfahren nach Anspruch 1, wobei das Verfahren mindestens eines der Folgenden detektiert und quantitativ bestimmt: (1) Dulcitol; (2) Dibromdulcitol; (3) eine andere Unreinheit als Dulcitol; und (4) ein Abbauprodukt von Dianhydrogalactitol.
  3. Verfahren nach Anspruch 1, wobei die Flussrate etwa 0,72 ml/min bis etwa 0,88 ml/min beträgt.
  4. Verfahren nach Anspruch 1, wobei das Elutionsschema wie folgt ist: (1) 0,00 Minuten, zu 90% 100%iges Acetonitril; zu 10% 100%iges Wasser; (2) 3,00 Minuten, zu 90% 100%iges Acetonitril; zu 10% 100%iges Wasser; (3) 20,00 Minuten, zu 75% 100%iges Acetonitril, zu 25% 100%iges Wasser; (4) 25,00 Minuten, zu 50% 100%iges Acetonitril, zu 50% 100%iges Wasser; (5) 30,00 Minuten, zu 50% 100%iges Acetonitril, zu 50% 100%iges Wasser; (6) 30,10 Minuten, zu 90% 100%iges Acetonitril, zu 10% 100%iges Wasser; und (7) 40,00 Minuten, zu 90% 100%iges Acetonitril, zu 10% 100%iges Wasser.
  5. Verfahren nach Anspruch 1, wobei das Injektionsvolumen etwa 15 µl bis etwa 35 µl beträgt.
  6. Verfahren nach Anspruch 1, wobei die Säulentemperatur etwa 28 °C bis etwa 32 °C beträgt.
  7. Verfahren nach Anspruch 1, wobei die Injektortemperatur etwa 20 °C bis etwa 22 °C beträgt.
  8. Verfahren nach Anspruch 1, wobei die Detektion mittels Verdampfungs-Lichtstreudetektion (ELSD) erfolgt.
  9. Verfahren nach Anspruch 8, wobei die Verdampfungs-Lichtstreudetektion den nach der Säule erfolgenden Zusatz eines flüchtigen Lösungsmittel beinhaltet, um das Verdampfen der wässrigen Fraktion einer vorhandenen mobilen Phase zu verbessern.
  10. Verfahren nach Anspruch 9, wobei das flüchtige Lösungsmittel aus der Gruppe ausgewählt ist, die aus Methanol, Ethanol, Isopropanol und Acetonitril besteht.
  11. Verfahren nach Anspruch 8, wobei die ELSD-Detektionsbedingungen Folgende sind: eine Temperatur von etwa 36 °C bis etwa 44 °C, ein Gasdruck von etwa 2,0 bar bis etwa 3,0 bar und eine Verstärkung von 7.
  12. Verfahren nach Anspruch 8, das ferner ein Elektrospray-Tandem-Massenspektrometer beinhaltet, das an das HPLC-System mit ELSD angeschlossen ist.
  13. Verfahren nach Anspruch 12, wobei die Massenspektroskopie in Tandem mit HPLC Molekülioneninformationen und mögliche chemische Strukturen, die eine Molmasse aufweisen, die mit den Molekülioneninformationen übereinstimmt, für jede der beobachteten Unreinheiten und Abbauprodukte bereitstellt.
  14. Verfahren nach Anspruch 12, wobei mindestens eine Unreinheit oder ein Abbauprodukt durch Trennung mittels Säulenchromatographie, gefolgt von mindestens einem Reinigungsverfahren zum Erhalten einer unbekannten Feststoffprobe, identifiziert wird.
  15. Analytisches Verfahren nach Anspruch 1, das ferner den Schritt des Durchführens einer präparativen HPLC-Erfassung von mindestens einem spezifischen Substanz-Peak, der in einer Zubereitung von Dianhydrogalactitol vorhanden ist, beinhaltet.
EP16869177.2A 2015-11-25 2016-11-22 Verfahren zur analyse und auflösung von präparaten von dianhydrogalactitol und derivaten oder analoga davon Active EP3380462B1 (de)

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